Sound waves let us see light deep within organs

Researchers stare into the depths of tissue using clever optics.

Taking pictures of the inside of the human body is something that we take for granted. But really, most current imaging systems are less than ideal. When it comes to getting the details without destroying tissues, there isn't much that beats modern optical imaging systems, especially fluorescent imaging systems. But the very thing that makes visible light so good for imaging also makes it impossible to image at any depth. As far as imaging goes, beauty really is only skin deep.

If there was some way to control how light was scattered through the intervening tissue, it would be possible to image much deeper. This is the challenge that has been taken up by a group of researchers at the Howard Hughes Medical Institute's Janelia Farm research center.

Your photons are ping-pong balls

You can't image deeply into tissue because the light bounces around like a ping-pong ball chased by a cat. Look at it this way: a cell, according to my high school biology text, contains an outer membrane, a fluid filled with tiny bubbles of stuff, a nucleus (surrounded by its own membrane), and DNA inside that.

We know this because when you shine light through a cell, a tiny amount of the light is reflected off all of these features. Since these surfaces are not flat (they are curved), the light that is reflected goes in many different directions. Likewise, some of the light that passes through has its direction changed by these surfaces (we call this refraction). The combination of these reflections and refractions is called scattering.

Now, for any single cell, only a tiny fraction of the light is scattered. But when imaging deeply through layers of cells, the cumulative effect is massive. Imagine that we are trying to capture something ten centimeters deep in the body. The light is going to have to pass through some 3000 cells to reach that depth. If each cell scatters one percent of the light then we can reasonably expect that only one of every 357×1012 photons will make it without scattering.

What this tells you is that, no matter what lens you use, that light simply isn't going to focus. Even worse, the information carried by the light exiting the body is a kind of sum of everything between the skin and the target depth. So, unless you already know what the object you are imaging looks like, you aren't going to get any pictures.

My photons are not ping-pong balls

Let's pretend for a moment that we had some magic method to identify light that had passed through the intended focal point of our lens, which is deep in a body. This light would not have gone straight to and from the focal point—each photon would have followed a random path. Since each photon has traveled a different distance, the electric fields of the photons are tangled up in a huge mess. This mess results in the light exiting the tissue in every direction.

We can't do anything about that—or at least we can't do anything about it without destroying the tissues we are trying to image. So instead, we carefully measure how the light exits the tissue. This information, called the wavefront, contains all the scattering information from the light. We can use that information to craft a light wave that, when sent into a body, will only travel along those scattering paths that pass through the intended focus.

This makes use of the idea that all the reflections and refractions are exactly reversible: if we send light in the opposite direction, it will follow exactly the same path back. The consequence is that if we can identify light that originates from a desirable location, we can make a light beam that will be focused on that location.

Stamping an identity on a photon

So far, somuchwater under the bridge. What makes this work unique and interesting is the way the researchrs have managed to identify photons that passed through an intended focal point.

The trick is to use ultrasound. Sound waves have a much longer wavelength than visible light. In a sense, the waves are too long to be very influenced by the structure of individual cells. Indeed, the refraction and reflection of sound waves is governed by changes in mechanical stiffness: transitions from tissue to bone, boundaries of organs, and such like. This allows ultrasound to be effectively focused deep in tissue.

When you do this, the tissue at the focus of the ultrasound wave is vibrated very vigorously by the sound waves. When visible light is scattered by the tissue at the focus, its frequency is increased or decreased by the frequency of the sound wave. In effect, the light that makes it to the tissue of interest is labelled.

Fluorescence imaging

In a lot of life sciences imaging, the natural contrast between different cellular features is not sufficient for good quality images. In cases like this, researchers often make use of fluorescence imaging to increase the contrast.

When a molecule absorbs a photon of energy, it enters an excited state. It has several ways to get rid of this energy: It can do this by movement, as it starts to vibrate and rotate, spreading energy through the environment as heat. It can also radiate another photon, and because this photon has less energy than the photon the molecule absorbed (since some was lost as heat), we can distinguish between the light emitted by the fluorescent molecule and the light used to excite the molecule.

These molecules can be designed so that they preferentially attach to certain sites on the cell. Some molecules like to sit in between the rungs of DNA, allowing DNA to be visualized. Others are designed to attach to the signaling molecules that stick out of cell membranes. More are designed to dock with certain proteins. In each case, the fluorescence tells us where and how much of the target is within the field of view of the imaging system.

After separating out and measuring the wavefront of the light that was scattered by the target of the sound wave, the researchers can imprint the reverse of that wavefront on a light beam traveling in the opposite direction. This light wave focuses at the same point that is the focus of the ultrasound wave.

We have focus, now, how do I make an image?

Now that we are able to focus the light to any location we desire, imaging is relatively simple. The laser is used to excite fluorescence in the tissue. Ordinarily, this might not work because fluorescence is emitted everywhere that the light passes. Thanks to the scattering, the exciting light field is very weak everywhere but the focus, so the vast majority of the fluorescence comes from the focus.

The light emitted by the fluorescent molecules is also scattered as it travels through the tissue, but in this case, we don't care. We simply collect as much of the light as we can and make a map of the intensity of this light as the excitation focus is scanned through the tissue.

Now, you might be thinking that relying on fluorescence makes this a pretty limited technique, but that would be wrong. There are plenty of molecules in the cell that are autofluorescent, so we can use those to image. But, more usefully, we can introduce fluorescent markers that selectively attach to molecules that are associated with specific tissues. An example might be to create a molecule that binds only to a particular form of cancer, or to insulin-producing beta cells.

This technique would then allow one to obtain images of these tissues. Another example is tracking the behavior of a drug. If one were to attach a fluorescent label to the drug, it becomes possible to monitor where it goes in the body, how long it takes to break down, andwhether it reaches the parts of the body where it is required.

I'm sold, but how good is it really?

This has a long way to go, however, before it is put to use outside the lab. The spatial resolution is about 40µm, which is basically given by the properties of the ultrasound pulse. But more importantly, this experiment takes an entire lab table of delicate optical equipment. To become some sort of useful clinical or research tool, it will have to go through a lot of development. Nevertheless, the essential parts are there, and it is now a matter of time and effort.

Chris Lee
Chris writes for Ars Technica's science section. A physicist by day and science writer by night, he specializes in quantum physics and optics. He lives and works in Eindhoven, the Netherlands. Emailchris.lee@arstechnica.com//Twitter@exMamaku

My physics is somewhat rusty - could someone remind me why the frequency at which the target is vibrating is added to or subtracted from the frequency of the light that passes through it?

Essentially, it's the Doppler Effect. If the tissue is oscillating, at some points in time it is moving towards the direction the light is now traveling, increasing frequency, and at other points it is moving away, decreasing frequency.

It's exactly like if a speaking on a pendulum were rocking towards/away from you while playing a constant tone, only way faster (and it obviously works better with it not moving in an arc, but rather a line).

Very cool, but it doesn't sound like it will be very useful for in-vivo imaging in people. If "the tissue at the focus of the ultrasound wave is vibrated very vigorously by the sound waves" followed by focusing so much laser energy on that tissue that it fluoresces, I don't want that done to *my* tissues, certainly not without a mountain of safety studies, that's for sure.

People are worried about a little low-frequency, low-intensity cell phone radiation; that kind of high intensity vibrating and zapping sounds like a recipe for *something* to get affected seriously.

Very cool, but it doesn't sound like it will be very useful for in-vivo imaging in people. If "the tissue at the focus of the ultrasound wave is vibrated very vigorously by the sound waves" followed by focusing so much laser energy on that tissue that it fluoresces, I don't want that done to *my* tissues, certainly not without a mountain of safety studies, that's for sure.

People are worried about a little low-frequency, low-intensity cell phone radiation; that kind of high intensity vibrating and zapping sounds like a recipe for *something* to get affected seriously.

If the choice is between dying because of ignorance or dying a little bit later because we can treat a known problem, I'll pick #2.

Very cool, but it doesn't sound like it will be very useful for in-vivo imaging in people. If "the tissue at the focus of the ultrasound wave is vibrated very vigorously by the sound waves" followed by focusing so much laser energy on that tissue that it fluoresces, I don't want that done to *my* tissues, certainly not without a mountain of safety studies, that's for sure.

People are worried about a little low-frequency, low-intensity cell phone radiation; that kind of high intensity vibrating and zapping sounds like a recipe for *something* to get affected seriously.

You do know we use ultrasounds to look at foetuses, right? Also, sound isn't a wave in the electromagnetic spectrum, so it's hardly fair to compare it to cell phone radiation.

Reading the article, you'll note the lasers frequently are used to fluoresce "fluorescent markers" designed to bind to specific cells. A practice ALSO currently done today.

You're right. I meant it wasn't electromagnetic radiation. The two are closely related, and I'm often lazy.

That's still not correct. Perhaps what you specifically have in mind is ionizing radiation (which still doesn't describe e.g. the output of cell radios, and is an entirely separate issue from electromagnetism of any form)?

You're right. I meant it wasn't electromagnetic radiation. The two are closely related, and I'm often lazy.

That's still not correct. Perhaps what you specifically have in mind is ionizing radiation (which still doesn't describe e.g. the output of cell radios, and is an entirely separate issue from electromagnetism of any form)?

Ok, ok, now I'm confused. Electromagnetic Radiation is "energy emitted...by charged particles." Sound is not a charged particle. It is the measure of oscillation through a medium (like air, steel, or flesh).

You're right. I meant it wasn't electromagnetic radiation. The two are closely related, and I'm often lazy.

That's still not correct. Perhaps what you specifically have in mind is ionizing radiation (which still doesn't describe e.g. the output of cell radios, and is an entirely separate issue from electromagnetism of any form)?

Ok, ok, now I'm confused. Electromagnetic Radiation is "energy emitted...by charged particles." Sound is not a charged particle. It is the measure of oscillation through a medium (like air, steel, or flesh).

Very cool, but it doesn't sound like it will be very useful for in-vivo imaging in people. If "the tissue at the focus of the ultrasound wave is vibrated very vigorously by the sound waves" followed by focusing so much laser energy on that tissue that it fluoresces, I don't want that done to *my* tissues, certainly not without a mountain of safety studies, that's for sure.

People are worried about a little low-frequency, low-intensity cell phone radiation; that kind of high intensity vibrating and zapping sounds like a recipe for *something* to get affected seriously.

You do know we use ultrasounds to look at foetuses, right? Also, sound isn't a wave in the electromagnetic spectrum, so it's hardly fair to compare it to cell phone radiation.

Reading the article, you'll note the lasers frequently are used to fluoresce "fluorescent markers" designed to bind to specific cells. A practice ALSO currently done today.

It sounds like the ultrasound they are using is a lot more intense than the normal imaging ultrasound, since it is highly focused on a very small area, and is used primarily for significant vibration of the tissues, rather than just timing reflection of small pulses from tissue boundaries, which is how normal ultrasound imaging works.

Possibly it is safe, but the burden is on them, and I'm skeptical that even the normal ultrasound will ultimately turn out to be totally safe. Such high-frequency (read: high energy) vibration of tissues seems like it *must* have the potential for damage to very small and delicate structural features. You can die from being shaken, this is just shaking on a microscopic level.

Come to think of it, ultrasound imaging started getting widespread use around 1963, and suddenly now we are finding people born since around then have much higher rates of autism. You never know, given how poor our medical industries' testing turns out to be, whether something like that might turn up. Not trying to be paranoid, but look how long we used BPA before finding out how bad it is.

As for the fluorescence, as far as I can tell that is primarily used on tissue samples, not in living human bodies. From glancing at the paper referenced below, it sounds like in-vivo use of deep-tissue fluorescence (using introduced fluorescent markers, which I think is what they are talking about here) is pretty much only for small lab animals. Quote: "there is no currently available NIR-emissive agent that possesses the ideal properties for human application" (meaning agents used for near-infrared fluorescent markers). Apparently nano-dots work, but are made from toxic materials.

You're right. I meant it wasn't electromagnetic radiation. The two are closely related, and I'm often lazy.

That's still not correct. Perhaps what you specifically have in mind is ionizing radiation (which still doesn't describe e.g. the output of cell radios, and is an entirely separate issue from electromagnetism of any form)?

Ok, ok, now I'm confused. Electromagnetic Radiation is "energy emitted...by charged particles." Sound is not a charged particle. It is the measure of oscillation through a medium (like air, steel, or flesh).

Sound itself isn't a charged particle, but it can be produced by one though when energy is converted

Sound is not a photon. Its not a carrier of the Electro Magnetic force. Sound does not have a spin. Sound does not travel at the speed of light. You can do a lot of cool stuff with sound but it behaves differently than radio waves, microwaves, IR, Visible Light, UV, etc...

Ultrasound, depending on its intensity, can damage tissue. In fact, this is one approach being explored as an alternative to surgery to remove tumours. The point is that, for imaging systems and such like, the total sound power sent into the body must satisfy be below a certain threshold. Likewise, laser light exposure is also regulated.

I do not know if the research here was within those limits (they use tissue phantoms), but, clearly, for clinical applications, they will have to keep the optical and acoustic intensities within the known limits for damage.

Very cool, but it doesn't sound like it will be very useful for in-vivo imaging in people. If "the tissue at the focus of the ultrasound wave is vibrated very vigorously by the sound waves" followed by focusing so much laser energy on that tissue that it fluoresces, I don't want that done to *my* tissues, certainly not without a mountain of safety studies, that's for sure.

People are worried about a little low-frequency, low-intensity cell phone radiation; that kind of high intensity vibrating and zapping sounds like a recipe for *something* to get affected seriously.

You do know we use ultrasounds to look at foetuses, right? Also, sound isn't a wave in the electromagnetic spectrum, so it's hardly fair to compare it to cell phone radiation.

Reading the article, you'll note the lasers frequently are used to fluoresce "fluorescent markers" designed to bind to specific cells. A practice ALSO currently done today.

It sounds like the ultrasound they are using is a lot more intense than the normal imaging ultrasound, since it is highly focused on a very small area, and is used primarily for significant vibration of the tissues, rather than just timing reflection of small pulses from tissue boundaries, which is how normal ultrasound imaging works.

Possibly it is safe, but the burden is on them, and I'm skeptical that even the normal ultrasound will ultimately turn out to be totally safe. Such high-frequency (read: high energy) vibration of tissues seems like it *must* have the potential for damage to very small and delicate structural features. You can die from being shaken, this is just shaking on a microscopic level.

Come to think of it, ultrasound imaging started getting widespread use around 1963, and suddenly now we are finding people born since around then have much higher rates of autism. You never know, given how poor our medical industries' testing turns out to be, whether something like that might turn up. Not trying to be paranoid, but look how long we used BPA before finding out how bad it is.

As for the fluorescence, as far as I can tell that is primarily used on tissue samples, not in living human bodies. From glancing at the paper referenced below, it sounds like in-vivo use of deep-tissue fluorescence (using introduced fluorescent markers, which I think is what they are talking about here) is pretty much only for small lab animals. Quote: "there is no currently available NIR-emissive agent that possesses the ideal properties for human application" (meaning agents used for near-infrared fluorescent markers). Apparently nano-dots work, but are made from toxic materials.